Cytokinins are a group of plant hormones that are powerful mitogens and which play a role in many plant developmental processes. An understanding of the molecular mechanisms by which cytokinins act could help to better understand the regulation of cell proliferation in human cells. Almost nothing definitive is known with regard to cytokinin signaling in higher plants. We propose to utilize an integrative approach to this problem, combining molecular, genetic and biochemical approaches to elucidate the elements involved in cytokinin action. The main thrusts of this proposal derive from our isolation of two cytokinin primary response genes, IBC6 and IBC7, which belong to a larger Arabidopsis gene family that is similar to bacterial signaling elements. We will exploit the cytokinin-specific induction of GFP fluorescence in an IBC6 promoter-GFP transgenic line to identify cytokinin-action mutants by identifying seedlings that inappropriately exhibit GFP expression. Preliminary screens indicate that this approach will be successful and that these mutants should help to dissect the cytokinin response pathway. The rapid induction of IBC6 and IBC7, coupled with their similarity to bacterial signaling elements, suggests that these genes may be involved in cytokinin signaling. We will test this hypothesis and other possible functions of this gene family by determining the effect of altered ARR gene expression, both gene disruption and regulated overexpression, on plant development and cytokinin responsiveness. We will also identify and characterize proteins that interact with IBC6 as a means of identifying potential downstream targets and upstream activators. We propose to identify the cis-acting DNA sequences responsible for the cytokinin-response of IBC6 as a means of starting to unravel the mechanisms by which cytokinins regulate gene expression. Once identified, we will use this element to identify additional potential cytokinin response genes and to identify DNA binding proteins responsible for the function of this element. Finally, we will use Affymetrix gene chips to identify novel cytokinin-regulated genes in Arabidopsis.